Project Summary The adaptive CD8+ T cell response is essential for controlling viral replication during acute and chronic stages of HIV infection and is a determining factor in HIV disease progression. However, despite induction of HIV-specific CD8+ T cell responses, infected individuals achieve only partial control of infection. The inability to eradicate HIV stems from the virus? ability to evade potent T cell responses via epitope mutation and driving T cell dysfunction. However, some persons are able to durably control viremia without the need for antiretroviral medications. While control is associated with ?protective? MHC-I alleles, it is not associated with the magnitude of CD8+ T cell responses as measured by standard IFN-? ELISPOT assays, but rather by functional ability to mediate particularly potent cytolytic function. As yet, no study has fully characterized the CD8+ T cell specificities that actually kill HIV-infected cells. Furthermore, previous work shows that CD8+ T cell cytokine effector function is discordant with cytolytic function. This, in part, is attributed to inefficient effector-target detachment, causing hyper-activation, and increased secretion of pro-inflammatory cytokines, which can contribute to inflammation, a hallmark of HIV infection. Given the importance of CD8+ T cells to cure and vaccine strategies, defining and engaging responses with optimal antiviral activity while limiting excessive T cell activation is of utmost importance. For the first part of this proposal I hypothesize that HIV epitope specificities of CD8+ T cells that kill rather than produce cytokine, an effect of perforin expression, better associate with and predict control of HIV infection. To address this hypothesis, two methods, one using perforin/IFN-? secretion as a readout for cytolytic/cytokine function, and the other, a novel microfluidics-based assay that couples single CD8+ T cells and targets, will be used to sort viable effector cells based on cytolytic or cytokine function. Transcriptional analysis of sorted populations will provide insight into pathways that regulate perforin and hence cytolytic function. In addition, perforin-based specificity mapping will compare HIV specificities of cytolytic CD8+ T cells for HIV+ progressors and controllers, providing insight into optimal antiviral responses to engage for potential therapeutics. For non-cytolytic effectors, which inefficiently detach from their target, limiting their excessive activation is essential to prevent unwanted downstream inflammation. Previous studies suggest that target cell apoptosis is required for effector-target detachment, yet the exact signal for detachment is unknown. Thus, for the second part of this proposal, I hypothesize that CTLs detach from target cells following recognition of phosphatidylserine (PS) exposure on the target, a common apoptotic signal. Immunological techniques will be used to probe the involvement of PS and PS-binding inhibitory proteins, such as Tim-3, in effector-target detachment. Manipulation of this signal would potentially provide a method to reduce the hyper-activation and subsequent inflammation caused by non-cytolytic effectors. In addition to HIV, these studies have implications for treatment of other immunopathogenic diseases, including Tuberculosis. !